1. We are studying the structure, function, and expression of the transmembrane channel-like (TMC) genes 1 and 2. We have successfully generated mice segregating knockout (null) alleles of Tmc1 and Tmc2. We are characterizing their mutant auditory phenotypes at the functional, cellular and molecular levels. We are using these mice to test the validity of staining patterns of antibodies to Tmc1 and Tmc2 in inner ear sensory hair cells. We have nearly completed delineating the transmembrane topologic organization of TMC1 protein expressed in tissue culture cells. Our results indicate that it is a six-pass transmembrane protein with cytoplasmically oriented N- and C-termini. We have used a yeast two-hybrid screen to isolate genes encoding proteins that potentially interact with Tmc1 and Tmc2. We have used a combination of approaches to determine which interactions might occur in situ in hair cells, and have narrowed the list to a few candidate genes implicated in vesicular trafficking and in antiapoptosis and cell survival. We have determined that background mouse strain has an effect on the rate and extent of outer hair cell degeneration in heterozygous Beethoven mice. We have genotyped the N2 offspring of a mapping backcross and identified the genetic map location(s) of at least four loci responsible for these differences. We are currently sequencing candidate genes in these modifier locus regions to identify the specific genetic changes that modify the loss of outer hair cells in Beethoven mice. In order to identify additional deafness-causing mutations of TMC1 that might lend insight into its function, we are performing a mutation analysis of TMC1 in 16 south Asian families segregating recessive deafness linked to markers near TMC1. We ascertained a large Caucasian family segregating nonsyndromic, autosomal dominant, postlingual progressive sensorineural hearing loss. A linkage screen revealed their deafness co-segregated with markers linked to TMC1. TMC1 sequence analysis demonstrated a missense substitution of histidine for aspartate at amino acid position 572. This is the same residue that is mutated in the only other known DFNA36 family that we reported three years ago. We are currently investigating the molecular basis for hearing loss caused by these mutations, which must act via a dominant negative or gain-of-function mechanism since heterozygous carriers of functional null (recessive) alleles of TMC1 have normal hearing. We have performed co-expression studies of wild type and mutant TMC1 which demonstrate that mutant and wild type polypeptides can associate with each other; it is possible that DFNA36 mutant allele products cause a dominant negative effect upon wild type TMC1 polypeptides by associating with them to form a nonfunctional complex. 2. We are generating knockout mice for Tmc3, Tmc6, and Tmc8 to better understand the function(s) of Tmc genes and proteins. Tmc3 appears to be expressed primarily in neuroendocrine tissues and organs. Tmc6 and Tmc8 are primarily expressed in lymphoid cells and tissues, and truncating allele of either TMC6 or TMC8 cause the autosomal recessive disease epidermodysplasia verruciformis, characterized by chronic cutaneous HPV infections and susceptibility to non-melanoma skin cancers, and defects in helper T cell function. We hav generated recominant ES cells for Tmc3 and Tmc6 knockouts and are expanding ES cell lines for injection into blastocysts. We have generated targeting constructs for Tmc8 and a Tmc6/Tmc8 double knockout (the genes lie next to each other on chr. 17) for electroporation into ES cells. 3. We are trying to positionally clone the gene mutaated in the mouse Twirler strain. Heterozygous Twirler mice have inner ear malformations and obesity, whereas homozygous mice are born with cleft palate and die at birth. The critical interval containing the Twirler gene is approximately 750 kilobases and contains one known gene and two predicted genes. We have completed nucleotide sequence analysis all of the exons in these genes and not found a pathogenic mutation. We have identified different splice isoform variants encoded by the Twirler allele of one of the predicted genes and we are carefully searching for mutations in the non-coding region of that gene. 4. We have completed our study of a genetic modifier of human hereditary hearing loss. This modifier is a missense variant of plasma membrane calcium pump PMCA2. We plan to test the hypothesis that this variant, or other variants of PMCA2, are causative factors for age-related hearing loss (presbycusis). 5. Enlargement of the vestibular aqueduct (EVA) is the most commonly detected radiologic malformation in temporal bones of individuals with hearing loss. A significant proportion of EVA cases have been reported to be associated with mutations of the SLC26A4 gene, in which mutations cause Pendred syndrome. PS is an autosomal recessive disorder comprised of bilateral sensorineural hearing loss and a defect in the ability of the thyroid gland to organify iodine. EVA is a universal finding in the ears of affected PS individuals. PS is correlated with two mutant SLC26A4 alleles, and nonsyndromic EVA is associated with one or zero mutant SLC26A4 alleles. Based upon our data, we hypothesize that one or more other genetic or environmental factors may act alone or in combination with a single SLC26A4 mutation to cause EVA. We have analyzed a number of candidate genes based upon known association with EVA in other hearing loss syndromes, or based upon molecular function, as candidates for the etiologic cofactor. We have not identified any convincing genetic cofactors by this approach, and are now actively accruing families with two or more individuals with EVA to perform an allele-sharing genomewide screen for such cofactors. We are also analyzing our genotype and phenotype data to identify clinical features that may guide molecular diagnosis or clinical prognosis. 6. We ascertained a large Caucasian family segregating autosomal dominant, nonsyndromic, postlingual-onset, progressive sensorineural hearing loss. We determined that their hearing loss was linked to markers at the DFNA10 locus. Nonsyndromic hearing loss DFNA10, as well as a syndromic form of hearing loss that includes dilated cardiomyopathy, are caused by truncating mutations of the EYA4 gene. We sequenced EYA4 and found that this family has a frameshift mutation. 7. We are completing a temporal bone histopathologic and genetic study of an infant with keratitis-ichthyosis-deafness syndrome. We have shown that this child was deaf due to developmental dysplasia of the cochlear epithelium associated with a mutation of GJB2, which encodes connexin 26. Our result indicates that at least some GJB2 mutations cause deafness by disrupting cochlear development.